Pegvisomant (Somavert®; Pharmacia Corp.) is a human growth hormone receptor antagonist. It is an analog of human growth hormone (“hGH”) that has been structurally altered. The amino acid sequence of the protein component/intermediate (B-2036) of pegvisomant differs from the amino acid sequence of hGH at nine positions. The specific amino acid substitutions are as follows: H18D, H21N, G120K, R167N, K168A, D171S, K172R, E174S, and 1179T. As is well recognized in the art, the first letter (i.e., H18D) represents the amino acid in the sequence of hGH at the numbered position (i.e., 18th amino acid position as indicated by H18D) which is substituted with the amino acid designated by the second letter (i.e., H18D). Therefore, H18D designates a substitution of the amino acid his by the amino acid asp at the 18th amino acid position of the wild-type hGH amino acid sequence.
FIG. 1A schematically shows the amino acid sequence structure of the protein component/intermediate (B-2036) of pegvisomant (PEG B-2036 or B-2036 PEG) with asterisks indicating the potential sites of polyethylene glycol polymer (“PEG” unit) attachment. Additionally, the amino acid sequence listing of the protein component/intermediate (B-2036-without PEG unit attachment) of pegvisomant is identified herein as SEQ. ID. NO. 1. For comparison, the amino acid sequence listing of human growth hormone is identified herein as SEQ. ID. NO. 2. Both sequence listings are provided herewith. See also Jorgensen et al., “Quantifying biosynthetic human growth hormone in Escherichia coli with electrophoresis under hydrophobic conditions,” J. Chromatography A 817:205-214 (1998) for the sequence of hGH.
Structurally, pegvisomant is a protein (containing 191 amino acid residues) to which predominantly 4 to 6 PEG units are covalently bound. The molecular weight of the protein component/intermediate (B-2036) of pegvisomant is 21,998 Daltons. The molecular weight of each PEG unit of pegvisomant is approximately 5000 Daltons. Thereby the predominant molecular weights of pegvisomant are approximately 42,000 (4 PEG units/molecule), 47,000 (5 PEG units/molecule) and 52,000 (6 PEG units/molecule) Daltons.
Referring to the agonist, and without being bound by theory, it is believed that endogenous hGH activates its receptors when a single hGH molecule binds to two of its adjacent (and identical) receptor molecules, inducing hormone-mediated receptor homodimerization. See U.S. Pat. Nos. 5,849,535 and 6,057,292. The activity of hGH depends on its ability to bind two of its adjacent (and identical) receptors across two separate binding sites (site 1 and site 2) on the same hGH molecule. These hGH binding sites, designated as site 1 and site 2, are numbered 1 and 2 to reflect the order of their binding to two adjacent (and identical) hGH receptors which mediate hGH-dependent homodimerization.
Further, without being bound by theory, it is believed that pegvisomant selectively binds to human growth hormone receptors (“GH receptors”) on cell surfaces, where it blocks the binding of endogenous human growth hormone, thereby interfering with human growth hormone signal transduction. The structural modifications to the protein portion (also called “component” or “intermediate”) of pegvisomant (relative to hGH) allow pegvisomant to competitively block interaction between an hGH molecule and an hGH receptor. Pegvisomant binds to the GH receptor, therefore, blocking GH binding since the receptor is occupied. The structural modifications prevent receptor dimerization, as a result signal transduction does not occur. By so blocking the required close interaction between an hGH molecule and an hGH receptor, pegvisomant blocks the hGH-mediated homodimerization of the hGH receptors, giving pegvisomant its antagonist activity.
This antagonist is used to treat conditions, including, but not limited to, acromegaly in patients who do not adequately respond to surgery, radiation therapy, and/or other conventional medical therapies, or who cannot otherwise tolerate these therapies. In addition, the structural modifications to the protein portion (B-2036) of pegvisomant cause it to exhibit a binding affinity for the prolactin receptor which is lower than that of hGH, thereby minimizing the undesirable lactation-related side effects associated with the use of pegvisomant.
The protein intermediate portion (B-2036) of pegvisomant is synthesized by a strain of Escherichia coli bacteria that has been genetically modified by the addition of a plasmid that carries a gene for the growth hormone receptor antagonist (B-2036). B-2036 is then recovered from the microbial cells and purified. The purified B-2036 is then pegylated to produce pegvisomant (PEG B-2036). U.S. Pat. Nos. 5,849,535 and 6,057,292 describe methods of making B-2036 and methods for conjugating one or more PEG units to B-2036, albeit without details as to how to decrease, reduce, eliminate, reverse and/or prevent the formation of unacceptably high levels of the trisulfide and des-phe isoform impurities thereof.
One of the problems encountered using conventional recombinant manufacturing methods to make B-2036 is the formation of its isoform impurities, such as its des-phe and the trisulfide isoforms. Another of the problems encountered using conventional manufacturing and purification methods to make B-2036 PEG (i.e., pegylated B-2036 such as pegvisomant) from B-2036 is the formation of an undesirable “aggregate” of B-2036 PEG as further discussed below.
The des-phe isoform impurity is one wherein the B-2036 molecule is missing its amino-terminal phenylalanine. See FIG. 1A depicting the subject amino-terminal phenylalanine residue (i.e., indicated by the letter “F”) adjacent the —NH2 end of B-2036. The trisulfide isoform impurity is one wherein the B-2036 molecule contains an extra sulfur atom that forms a “trisulfide bridge” within the molecule. See box in FIG. 1B. Also, see Andersson et al., “Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli,” Int. J. Peptide Protein Res. 47:311-321 (1996) and A. Jesperson et al., “Characterisation of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli,” Eur. J. Biochem. 219:365-373 (1994). Without being bound by theory, it is believed that these isoform impurities typically are generated during cell growth (e.g., fermentation) and expression (synthesis and secretion) of B-2036 in genetically modified host cells, and/or during extraction and purification of the B-2036 protein.
Regarding the problem with the “aggregate,” formation of such “aggregate” leads to a decreased yield of the desired protein and to an increased cost of producing the same. Also, if the “aggregate” level is too high, the final protein may be of such low purity that it becomes unsuitable for therapeutic use.
Regarding certain impurities, International Application WO 94/24157 (published Oct. 27, 1994) discloses a hydrophobic derivative of hGH comprising an extra sulfur atom as compared to the native hGH. See WO 94/24157 at page 3, lines 3-10. The extra sulfur atom of the hydrophobic derivative of hGH forms a “trisulfide bridge” yielding an hGH trisulfide variant. See WO 94/24157 at page 7, lines 11-16. The WO 94/24157 reference further states that this hGH trisulfide variant can be converted back to its native hGH form by treating the hGH trisulfide variant with a mercapto compound such as cysteine, glutathione, 2-mercapto ethanol or dithiothreitol. See WO 94/24157 at pages 4 and 5.
International Application WO 96/02570 (published Feb. 1, 1996) describes another method for converting the hGH trisulfide variant back to its native form using either sodium sulfite, potassium sulfite, ammonium sulfite, or an alkaline-earth metal sulfite such as magnesium sulfite or calcium sulfite. See WO 94/24157 at page 4, lines 17-21.
International Application WO 00/02900 (published Jan. 20, 2000) entitled “Method for the production of recombinant peptides with a low amount of trisulfides” discusses “a method for the reduction of the amount of trisulfides in the production of recombinant peptides, e.g., both proteins and smaller peptides. The invention is based on the novel and unexpected finding that the amount of trisulfides in the production of recombinant peptides could be reduced by the addition of a metal salt, preferably in excess, already during or after fermentation and not, as earlier suggested, by conversion of the formed trisulfides of growth hormone into the native form.” See WO 00/02900 at page 2, lines 21-27. The WO 00/02900 reference further states “[t]he protein can be any recombinant protein but is preferably recombinant growth hormone which can be both human and animal such as human growth hormone (hGH), bovine growth hormone (bGH) and porcine growth hormone (pGH).” See WO 00/02900 at page 3, lines 4-6.
International Application No. WO 02/057478 (published Jul. 25, 2002) entitled “Methods and Composition For Extracting Proteins From Cells” is directed to a method of releasing a protein from a host cell by contacting the host cell with a reducing agent and a detergent. The reference states that the purpose of the reducing agent is to “facilitate[ ] the recovery of proteins in their native conformations.” See WO 02/057478 at page 2, lines 16-18. Furthermore, WO 02/057478 describes that the “one or more reducing agents are agents . . . that reduce disulfide bonds and/or maintain sulfhydryl residues in the[ir] reduced form. Any such reducing agent or agents may be used. In a preferred embodiment, the one or more reducing agents used are selected from the group consisting of, dithiothrietol (DTT); dithioerythritol (DTE); Cysteine (Cys) and Tris 2-carboxyethyphosphine (TCEP).” See WO 02/057478 from page 3, line 24 to page 4, line 4.
For other references regarding purification see U.S. Pat. No. 6,265,542 B1 (Fahrner et al. entitled “Purification of Molecules”); U.S. Pat. No. 6,333,398 B1 (Blank entitled “Protein Purification”); U.S. Pat. No. 5,747,639 (Seely entitled “Use of Hydrophobic Interaction Chromatography to Purify Polyethylene Glycols”); International Application No. PCT/US96/19459 (Ibrahim et al. entitled “Activated Linkers and Methods for Making and Purifying the Same”); and U.S. Patent Application No. U.S. 2002/002271 A1 (Rinderknecht et al. entitled “Antibody Purification”).
The above-noted references, however, are silent with regard to the prevention, reversal, reduction, or elimination of isoform impurity formation associated with a growth hormone antagonist such as pegvisomant or its protein portion, B-2036 and/or aggregate formation of pegylated protein, e.g., pegvisomant. Accordingly, there is a need for improved methods of making B-2036 that decrease, attenuate, prevent, minimize, reverse and/or eliminate the formation of its isoform impurities (trisulfide and/or des-phe) and/or aggregate formation of pegylated protein. Likewise, these references also are silent as to the detection, attenuation, minimization, reversal, reduction or elimination of the formation of the des-phe isoform impurity of growth hormone and/or aggregate formation of pegylated protein, e.g., pegylated growth hormone or pegylated human growth hormone. Accordingly, there is a need for improved methods of making growth hormone that decrease, attenuate, prevent, minimize, reverse and/or eliminate the formation of its des-phe isoform impurity and/or aggregate formation of pegylated protein, e.g., pegylated growth hormone or pegylated human growth hormone.